Visual evaluation method of gold cluster fluorescent probe on hematopoietic system injury caused by radiation
By modifying specific markers on the gold cluster fluorescent probe and combining infrared two-zone fluorescence imaging technology, the problems of insufficient sensitivity and difficulty in imaging evaluation of radiation damage hematopoietic system in the prior art are solved, and targeted imaging and high-resolution evaluation of hematopoietic stem cells are achieved.
Patent Information
- Application Number
- CN202411902393.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-04-11
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to achieve early, accurate and non-invasive assessment of radiation-induced hematopoietic system damage, and traditional imaging methods are difficult to achieve high resolution and deep imaging effects.
A method based on gold cluster fluorescent probe is developed to achieve targeted imaging of hematopoietic stem cells by modifying specific markers, such as anti-CD34, on the surface of the probe, and high-resolution, deep-level dynamic imaging is performed using infrared two-zone fluorescence imaging technology.
Real-time, accurate and non-invasive assessment of the hematopoietic system of radiation damage is achieved, meeting the clinical needs of early diagnosis and efficacy monitoring, and providing high-quality visual evaluation methods.
Smart Images
Figure HDA0005203545050000011 
Figure HDA0005203545050000012 
Figure HDA0005203545050000021
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedical imaging and radiation damage detection, and specifically relates to a method for visualizing the damage of the hematopoietic system caused by radiation using a gold cluster fluorescent probe, which can realize targeted imaging of hematopoietic stem cells in vivo. Background Art
[0002] In contemporary society, the risk of radiation exposure faced by the public has increased significantly, and such risks may come from radiotherapy, X-ray diagnosis and radiation contamination. These radiation exposure factors will cause varying degrees of damage to the hematopoietic system, which in turn will cause damage to the hematopoietic function and immune system, and severe cases may threaten life. Therefore, early and accurate assessment of radiation-induced hematopoietic system damage is of great significance for disease diagnosis and treatment. Hematopoietic stem cells are the functional core of the hematopoietic system, and their self-renewal and differentiation abilities provide continuous support for blood homeostasis. Hematopoietic stem cell migration imaging can be used to assess the degree of hematopoietic system damage, which is of great significance for the diagnosis and treatment monitoring of hematopoietic system damage caused by radiation and other factors. Traditional imaging methods, such as magnetic resonance imaging (MRI), computer-assisted tomography (CT), positron emission tomography (PET), etc., are difficult to achieve imaging detection of hematopoietic stem cells or the resolution does not meet the requirements. Optical imaging combined with fluorescent probes can achieve in vivo quantitative detection of hematopoietic stem cells, but the depth of tissue penetration is limited.
[0003] In recent years, NIR-II (wavelength range 1000-3000nm) fluorescence imaging technology has gradually become a research focus in the field of biomedical imaging due to its strong tissue penetration ability, low background noise and high-resolution imaging. As an innovative nanomaterial, gold cluster fluorescent probes exhibit excellent optical properties and biocompatibility in the second infrared region. By modifying their surfaces, they can achieve targeted recognition of specific cells. Therefore, combining NIR-II fluorescence imaging technology with gold cluster probes is expected to break through the limitations of traditional imaging technology and provide a new tool and method for in vivo monitoring of hematopoietic stem cells.
[0004] Therefore, technicians in this field are eager to develop a fluorescent probe for gold clusters in the second infrared zone, aiming to achieve visual evaluation of radiation-induced hematopoietic system damage. This probe will have the ability to accurately target and image hematopoietic stem cells, while achieving high-resolution, deep-level dynamic imaging effects in vivo. This method will provide a high-quality visual evaluation method, laying an experimental foundation for the diagnosis and treatment monitoring of hematopoietic system damage. It is of great significance in the basic research of radiation-induced body damage, especially hematopoietic system damage and treatment, as well as clinical precision and personalized diagnosis and treatment. Summary of the invention
[0005] The present invention aims to develop a visual evaluation method for radiation-induced hematopoietic system damage based on gold cluster fluorescent probes to solve the problems of insufficient sensitivity, difficulty in dynamic monitoring, and strong invasiveness in the existing radiation damage evaluation methods. Through the highly biocompatible and highly specific gold cluster fluorescent probes, real-time, accurate, and non-invasive evaluation of hematopoietic system damage can be achieved to meet the clinical needs of early diagnosis of radiation damage and efficacy monitoring.
[0006] The technical purpose of the present invention is to realize a visual evaluation method of radiation-induced hematopoietic system damage through the following technical scheme:
[0007] A visual evaluation method for radiation-induced hematopoietic system damage using a gold cluster fluorescent probe.
[0008] As a limitation of the above method, the in vivo evaluation of radiation-induced hematopoietic system damage specifically refers to real-time imaging monitoring of hematopoietic stem cells for damage to hematopoietic stem cells caused by radiation damage, and evaluating the transplantation effect based on this.
[0009] As a limitation of the above method, the surface of the gold cluster fluorescent probe has specific markers, including but not limited to one or more of anti-CD34, CD133 (Prominin-1), CD90 (Thy-1), CD117 (c-Kit), CD73, CD105 (Endoglin), CD44, CD45 and other stem cell marker antibodies.
[0010] As a limitation of the above method, the gold cluster fluorescent probe has good biocompatibility, low toxicity and high stability under physiological conditions.
[0011] As a limitation of the above method, the gold cluster probe can realize infrared zone II in vivo imaging with high sensitivity and high resolution, and can be used for precise positioning and quantitative analysis of radiation damage sites.
[0012] As a limitation of the above method, the gold cluster probe can achieve specific binding to mouse hematopoietic stem cells.
[0013] As a limitation of the above method, the gold cluster probe can achieve specific binding to human hematopoietic stem cells.
[0014] As a limitation of the above-mentioned method, the method can be further used for dynamic monitoring and evaluation of the therapeutic effect of radiation injury.
[0015] Based on the above technical scheme, the present invention aims to provide a targeted gold cluster fluorescent probe visualization evaluation method for radiation-induced damage to the hematopoietic system. This method can evaluate the damage of hematopoietic stem cells caused by radiation and the effect of their transplantation, thereby demonstrating important application value and significance for hematopoietic stem cell transplantation in the treatment of blood system diseases and immunodeficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:
[0017] Figure 1 This is a study on the specificity of targeted gold cluster fluorescent probes for mouse hematopoietic stem cells.
[0018] Figure 2 This is a study on the specificity of targeted gold cluster fluorescent probes for human hematopoietic stem cells.
[0019] Figure 3 Fluorescence imaging of various organs in mice after tail vein injection of targeted gold cluster fluorescent probe.
[0020] Figure 4 This is a study on the targeting of hematopoietic stem cells by targeted gold cluster fluorescent probes at different time points in vivo, and fluorescence imaging of the legs at different time points after radiation damage.
[0021] Figure 5 These are the infrared zone II fluorescence signals of mouse legs at different time points after mouse hematopoietic stem cell transplantation and human hematopoietic stem cell transplantation. DETAILED DESCRIPTION
[0022] Those skilled in the art can achieve the corresponding technical optimization goals by reasonably adjusting the process parameters according to the contents described herein. It is particularly pointed out here that all similar alternatives and improvement measures are obvious to those skilled in the art and should be regarded as included in the protection scope of the present invention. The method and application of the present invention have been comprehensively and deeply explained through preferred embodiments. It is obvious that relevant personnel can make necessary adjustments, appropriate modifications or reasonable integration of the method and application without violating the core content and protection scope of the present invention to realize and apply the technical solution of the present invention.
[0023] In order to more clearly and accurately explain the objectives, technical solutions and advantages of the present invention, the present invention will be comprehensively and thoroughly explained below in combination with specific implementation cases and drawings of the specification. The experimental methods used in the implementation cases are all performed in accordance with conventional means unless otherwise explicitly stated; the materials, reagents, methods and instruments and equipment used are also, unless otherwise explicitly stated, within the conventional scope of the technical field and can be obtained through regular commercial channels in the technical field.
[0024] The sources of raw materials and instrument models used in the examples are described below.
[0025] Figure 1 and Figure 2 The specificity research results presented are based on the infrared two-zone fluorescence microscope of Shunyu Instruments.
[0026] Figure 3 , Figure 4 and Figure 5 The results of near-infrared zone II optical imaging presented here were obtained with the help of an imaging system independently developed and constructed. The core components of the system include a NIRvana:640ST near-infrared zone II camera produced by Princeton Instruments, USA. At the same time, the system also integrates a FLC300-0808-8W-F200 laser produced by Frankfurt Laser Company, Germany. Relying on these high-precision equipment and carefully planned optical path layout, the researchers were able to successfully carry out high-quality near-infrared zone II optical imaging experiments.
[0027] Figure 3 , Figure 4 as well as Figure 5 The C57 male mice (6-8 weeks old) used in the in vivo imaging experiments presented in the present invention were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
[0028] The reagents and process equipment used in the present invention are commonly used in the prior art and are not critical to the present invention, so they are briefly described.
[0029] Embodiment 1
[0030] Targeting of the targeted gold cluster fluorescent probe to mouse hematopoietic stem cells:
[0031] The extracted mouse hematopoietic stem cells were cultured in vitro, smeared, and incubated with the targeted gold cluster fluorescent probe for 2 hours. After DAPI counterstaining, the binding of the targeted gold cluster fluorescent probe to the hematopoietic stem cells was observed under 808nm laser excitation by near-infrared microscopy to investigate the near-infrared imaging effect and evaluate the targeting of the targeted gold cluster fluorescent probe to mouse hematopoietic stem cells. Figure 1 It can be seen that when the targeted gold cluster fluorescent probe is co-incubated with the target cells, it exhibits a clear and obvious fluorescent signal, indicating its specific binding ability to hematopoietic stem cells. The experiment was repeated three times and the same results were obtained. This specificity is essential for achieving high-quality imaging and accurate tracking of hematopoietic stem cells. The specificity of this fluorescent probe ensures minimal interference from non-target cells and background signals, thereby improving imaging quality and accuracy. Therefore, the targeted gold cluster fluorescent probe has great potential in promoting research and application in the field of hematopoietic stem cell imaging.
[0032] Embodiment 2
[0033] Targeted gold cluster fluorescent probe targeting human hematopoietic stem cells:
[0034] Human hematopoietic stem cells were cultured in vitro, smeared, and incubated with targeted gold cluster fluorescent probes for 2 hours. After DAPI counterstaining, the binding of the developer molecules to human hematopoietic stem cells was observed under 808nm laser excitation by near-infrared microscopy to investigate the near-infrared imaging effect and evaluate the targeting of targeted gold cluster fluorescent probes to human hematopoietic stem cells. Figure 2 It can be seen that when the targeted gold cluster fluorescent probe is co-incubated with the target cells, it shows a clear and obvious fluorescent signal, indicating that it has good specific binding ability to human hematopoietic stem cells. The experiment was repeated three times and the same results were obtained. The specific targeting ability of the targeted gold cluster fluorescent probe to human hematopoietic stem cells lays the foundation for studying human hematopoietic stem cells, understanding their in vivo behavior, and developing new monitoring measures for hematopoietic stem cell-related therapies.
[0035] Embodiment 3
[0036] Distribution of targeted gold cluster fluorescent probes in mouse organs:
[0037] 200 μL of targeted gold cluster fluorescent probe was injected into 6-8 week old C57 male mice through tail vein. After 12 hours, the organs were dissected and the organs of the mice were placed under near infrared imaging equipment. Fluorescent signals were clearly observed in the femur, tibia and spine of the mice, such as Figure 3 As shown. It can be further proved that the targeted gold cluster fluorescent probe can accurately target hematopoietic stem cells.
[0038] Embodiment 4
[0039] Visual Monitoring of Murine Hematopoietic Stem Cell Transplantation:
[0040] In order to obtain mouse hematopoietic stem cells, the experiment selected C57 mice, took their femurs, and after muscle tissue stripping, used DMEM medium containing 10% fetal bovine serum (FBS) for bone marrow flushing. Subsequently, the collected bone marrow cells were filtered through a 300-mesh filter into a 50 mL centrifuge tube, the solution volume was adjusted to 12 mL, and an equal volume of room temperature lymphocyte separation solution was added. The centrifugal operation parameters were set to a temperature of 20 ° C, a speed of 1400 rpm, and both the speed of increase and the speed of decrease were set to zero, and the centrifugation time was 20 min. After the centrifugation was completed, the upper clear DMEM solution was first removed with a pipette, and then the cloudy cell layer in the middle of the solution (mainly bone marrow mononuclear cells, BM-MNCs) was slowly and circularly aspirated and transferred to a new centrifuge tube. Then, the cells were washed twice with DMEM medium containing 10% FBS (centrifugation conditions: temperature 4 ° C, speed 1200 rpm), and finally a bone marrow mononuclear cell population containing hematopoietic stem cells was obtained.
[0041] The extracted hematopoietic stem cells are precisely injected into the recipient mice whose original hematopoietic system has lost function due to radiation damage by tail vein injection. The number of injected cells is controlled within the range of 10^5 to 10^6, and during the injection process, it is strictly necessary to ensure that no bubbles are generated to reduce cell loss and minimize damage to the mice. After completing the injection and waiting for 15 minutes, the mouse live imaging experiment can be carried out. For specific operation steps, see Example 5.
[0042] Embodiment 5
[0043] In vivo imaging of hematopoietic stem cells in mice:
[0044] Different counts of allogeneic mouse hematopoietic stem cells were injected into the tail vein of the model mice that had been damaged by radiation. The syringe was held in the right hand and pierced into the abdominal cavity at a 45° angle on the left side of the lower abdomen of the mouse. 2% tribromoethanol (dose of 0.1 ml / 10 g) was injected to anesthetize the mouse. While the mouse was anesthetized, the mouse was depilated using a small animal depilatory. The skin was exposed to the air, which was conducive to our observation of the fluorescence distribution in the body. 200 μL of gold cluster fluorescent probe was injected into the mouse through the tail vein after depilation, and the mouse was fixed on the infrared zone II camera stand. Subsequently, under the excitation of 808 nm laser and the action of a 1000 nm long-pass filter, the infrared zone II camera detector can receive the infrared zone II fluorescence emitted by the fluorescent probe. The mouse femur was monitored at different times, and the fluorescence signal was recorded by a near-infrared camera. During the imaging process, the mouse was anesthetized by inhalation gas anesthesia using isoflurane to ensure that the body position remained unchanged during the imaging process and the imaging effect was not affected. Figure 4 and Figure 5As shown in the figure, in mice that have been damaged by radiation, without mouse or human hematopoietic stem cell transplantation, the fluorescence signal at the femur of the mouse gradually decays as the body's metabolism proceeds until it disappears. However, the femur of mice that have undergone mouse and human transplantation can maintain the fluorescence signal for a longer time. Therefore, by evaluating the intensity of the fluorescence signal, it is possible to detect whether the hematopoietic stem cells in the femur of the mouse are successfully transplanted.
[0045] The present invention mainly focuses on a method for visually evaluating radiation-induced hematopoietic system damage using targeted gold cluster fluorescent probes. Through in vitro cell experiments, this method verifies that the gold cluster probe can accurately target mouse and human hematopoietic stem cells. At the same time, the organ distribution of the targeted probe in the animal body was verified, further confirming its targeting performance. Subsequently, after 6-8 week-old C57 male mice received radiation damage, tail vein transplantation of mouse and human hematopoietic stem cells was implemented. After the transplantation was successful, the targeted gold cluster fluorescent probe was injected through the tail vein, and in vivo imaging was performed using a near-infrared camera. The experimental results showed that the legs of mice that had successfully transplanted mouse and human hematopoietic stem cells showed bright fluorescent signals, in sharp contrast to non-transplanted mice, thereby verifying the success of hematopoietic stem cell transplantation. The present invention fully demonstrates the significant potential of targeted gold cluster fluorescent probes in the field of animal hematopoietic stem cell transplantation imaging and evaluation.
[0046] The contents not elaborated in the specification of the present invention belong to the technical scope generally understood and mastered by those skilled in the art. Although the present invention has been disclosed through the above-mentioned preferred embodiments, this is not intended to limit the protection scope of the present invention. Any person familiar with the technology in this field can make appropriate adjustments and improvements to the present invention without deviating from the core idea of the present invention and the protection scope defined therein. Therefore, the protection scope of the present invention shall be based on the contents clearly recorded in the claims.
Claims
1. A visual evaluation method for radiation-induced hematopoietic system damage using a gold cluster fluorescent probe.
2. The method according to claim 1, characterized in that The method can be used to evaluate in vivo mice with hematopoietic system damage caused by radiation, specifically, to perform real-time imaging monitoring of hematopoietic stem cells for damage to hematopoietic stem cells caused by radiation damage, and to characterize the number of hematopoietic stem cells and the degree of hematopoietic system damage accordingly.
3. The method according to claim 1, characterized in that The surface of the gold cluster fluorescent probe has specific targeting molecules, including but not limited to one or more of anti-CD34, CD133 (Prominin-1), CD90 (Thy-1), CD117 (c-Kit), CD73, CD105 (Endoglin), CD44, CD45 and other stem cell marker antibodies.
4. The method according to claim 1, characterized in that: The gold cluster fluorescent probe has good biocompatibility, low toxicity and high stability under physiological conditions.
5. The method according to claim 1, characterized in that The gold cluster probe can achieve in vivo imaging in the second infrared zone with high sensitivity and high resolution, and can be used for precise positioning and quantitative analysis of radiation damage sites.
6. The method according to claim 1, characterized in that Gold cluster probes can achieve targeting of mouse hematopoietic stem cells.
7. The method according to claim 1, characterized in that Gold cluster probes can achieve targeting of human hematopoietic stem cells.
8. The method according to claim 3, characterized in that This method can be further used for dynamic monitoring and evaluation of the therapeutic effects of radiation injury.